Background: Urospermum picroidesis an invasive medicinal plant well established in North cost, Egypt. Endophytic fungi of this medicinal plant in Egypt is poorly known. Aim: The present study aimed to estimate the biological activities of endophytic fungi from Egyptian U.picroides. Materials and Mthods: Two endophytic fungi were isolated from U.picroides and identified as Fusarium oxysporum and Phoma herbarum based on phylogenetic analysis. Results: Ethyl acetate extracts of F. oxysporum and P. herbarum gave high antioxidant activities (26.2 and 51.7%, respectively). Moreover, they showed highly antitumor activities with some significant morphological changes of characteristic apoptosis accompanied by up regulation of both p53 and Bax for F.oxysporum and P.herbarum extracts (1.55, 2.14 for p53 and 1.24, 2.2 for Bax, respectively) with down regulation of Bcl-2 (0.18 and 0.16, respectively). Ethyl acetate extract of F.oxysporum (EAFE) showed highest antimicrobial activity against Trichophyton mentagrophytes (70 mm), while ethyl acetate extract of P.herbarum (EAPE) showed highest antimicrobial activity against Bacillus cereus (37 mm) and Candida albicans (35 mm). Scanning electron microscope micrographs showed major abnormalities for tested microorganisms after treatment with (EAFE and EAPE) resulting in complete alternation in their morphology. GC-MS results showed 30 biological active compounds for both extracts. The most significant proportion in EAFE was Diisooctyl phthalate with relative levels (74.9%), while Benzoic acid, 3, 5-bis (1, 1-dimethylethyl)-4-hydroxy- was the most active compound in EAPE (61.7%). Conclusion: These results proposed that ethyl acetate extracts of endophytic fungi from Egyptian U.picroides showed to be promising novel antioxidant, antitumor agents and antimicrobial with further phytochemical studies.